PROJECT SUMMARY NMDA receptors (NMDARs) are neurotransmitter receptors found at nearly all vertebrate excitatory synapses and contribute to nearly all nervous system functions. Ca2+ influx through NMDARs regulates critical neuronal functions such as synaptic plasticity and the slow signaling kinetics of NMDARs greatly impact the integration of postsynaptic signals. Abnormal NMDAR activity is involved in a remarkable range of nervous system disorders including schizophrenia, major depressive disorder, stroke, neuropathic pain, and neurodegenerative diseases. Memantine (Mem) and ketamine (Ket) are two clinically useful NMDAR open channel blockers, antagonists that prevent ion flux by binding in and physically occluding the NMDAR channel. Despite their similar basic mechanisms of action and pharmacological properties, Mem and Ket display surprisingly disparate clinical effects. This proposal aims to investigate a major biophysical difference between how Mem and Ket affect NMDAR function that may contribute to the divergent effects of Mem and Ket on brain function. We recently discovered that inhibition of the GluN1/2A subtype of NMDAR by Mem, but not by Ket, depends on intracellular Ca2+ concentration ([Ca2+i]). Our data suggest that Ca2+i-dependent channel block (CDB) by Mem is due to Mem stabilizing a Ca2+-dependent desensitized state of the receptor, implying that Mem and Ket may differentially alter the timing of synaptic responses. Furthermore, Mem CDB appears to depend on NMDAR subtype, which varies by subcellular localization, developmental stage, and cell type. Thus, CDB may allow Mem to target different subpopulations of NMDARs than Ket and therefore could contribute greatly to the differential effects of Mem and Ket. However, the mechanisms underpinning Mem CDB are currently unknown, preventing further investigation of the role of CDB in the effects of Mem on brain function. The central goal of this proposal is to identify the mechanism and structural determinants underlying CDB by testing the hypothesis that Mem CDB results from the stabilization of a Ca2+-dependent desensitized receptor state by Mem. The long-term goals of this proposal are a) to better understand NMDAR-channel blocker interactions to identify features of beneficial drugs and b) to incorporate models of open channel blockers into models of neuronal circuit function. Electrophysiological recordings from cells modified to express a specific NMDAR subtype will be used to thoroughly characterize and identify the structural underpinnings of CDB. Data from these experiments will be incorporated into kinetic and molecular models of NMDAR-channel blocker interactions, which will then be validated and refined with additional experiments. This iterative combination of modeling and experimentation will aid our mechanistic understanding of how Mem inhibition is regulated by intracellular Ca2+. Findings from this proposal will have broad translational potential, aiding in the design of more clinically efficacious NMDAR antagonists, and will deepen our understanding of basic NMDAR function.